Prions are infectious proteins that cause devastating neurodegenerative diseases (transmissible encephalopathies) in humans, cattle and other mammals. A puzzling aspect of these proteins is that they form not only a single 2-sheet rich conformation, but a suite of related aggregated conformers that are responsible for different prion diseases. Naturally occurring prions in yeast also display very similar behavior. A key aspect of different prion strains in both mammals and yeast is that they have unique capacities for traversing species barriers, which is poorly understood but has broad biological importance. The goal of this proposal is to investigate how differences in the structure of infectious prion conformers govern their ability to establish and overcome species barriers. Unfortunately, it is currently not possible to form highly infectious prions from recombinant mammalian prion protein (PrP) for structural studies, but this is readily possible for yeast prions. Therefore, we propose to investigate the structural relationship between prion strains and species barriers for the yeast prion Sup35 from S. cerevisiae (Sc) and C. albicans (Ca). A robust prion species barrier exists between Sc and Ca Sup35, but a Sup35 chimera composed of domains from both species can overcome this barrier. Using peptide microarrays we discovered that the capacity of chimeric prions to overcome this barrier is encoded by two small elements of primary sequence (named "recognition elements"), one from each yeast species (Tessier &Lindquist, Nature, 2007). The key hypothesis we propose to test is that the species-specific infectivities of two different prion strains are due to differences in the location of the prion recognition elements relative to the amyloid core. For the chimeric strain specific for infecting S. cerevisiae, we hypothesize that the Sc Sup35 recognition element is within the amyloid core (solvent shielded) while the Ca Sup35 recognition element is either outside the amyloid core (solvent exposed) or in an incompetent, non-amyloid conformation (weakly solvent shielded), and vice versa for the other strain conformation. We propose to test this hypothesis through the following three specific aims: 1) To generate single cysteine mutants in the prion domain of the Sup35 chimera in both bacterial and yeast expression plasmids, 2) To evaluate if single cysteine mutants have the same prion properties as the wild-type chimera by characterizing their prion behavior both in vitro and in vivo, and 3) To determine the degree of solvent exposure of cysteines in each strain conformation of the chimeric prion. The data obtained in this study will provide important structural information on how the locations of key amino acid sequences relative to the prion amyloid core impact the capacities of prions to overcome species barriers. Moreover, these findings will inform investigations of mammalian prions by providing testable hypotheses of how different mammalian prion strains traverse species barriers. PUBLIC HEALTH RELEVANCE: The transmission of infectious prions from cattle to humans continues to threaten our society. Prions form multiple infectious strain conformations that have unique capacities to overcome species barriers through an unknown mechanism. This proposed research aims to decipher this mechanism with the long-term goal of rationally developing therapeutics to prevent and reverse interspecies prion transmission.